﻿Baiyuerius gen. nov., a new genus of Coelotinae (Araneae, Agelenidae) spiders from China and Vietnam

﻿Abstract Baiyueriusgen. nov., a new genus of the subfamily Coelotinae F. O. Pickard-Cambridge, 1893 is described, including five new species: B.daxisp. nov. (♀), B.pindongsp. nov. (♂), B.tamdaosp. nov. (♀), B.zhupingsp. nov. (♂) and B.zuojiangsp. nov. (♂♀), from southern China and northern Vietnam. Our molecular phylogenetic analyses support Baiyuerius gen. nov. as monophyletic and as a sister group of the newly established genus Yunguirius Li, Zhao & Li, 2023.

Upon examination of specimens collected from southern China and northern Vietnam, we suspected that they should belong to a new genus and five undescribed putative species. Therefore, morphological and phylogenetic analyses and comparisons with closely related species were carried out to confirm this. Here we report the results of these analyses, and describe in detail the new species and the genus erected to accommodate them.

Sampling and morphological examination
All specimens studied in this paper were collected from southern China and northern Vietnam and are deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS). Specimens were examined with a LEICA M205 C stereomicroscope at IZCAS. Photos were taken with an Olympus C7070 wide zoom digital camera (7.1 megapixels) mounted either on an Olympus SZX12 dissecting microscope or on an Olympus BX51 compound microscope. Images from multiple focal ranges were combined using Helicon Focus v.6.80 photo stacking software. The epigyne and male palp were dissected for examination. The epigyne was treated in a warm 10% potassium hydroxide (KOH) solution. Images of the left male palp are illustrated. Measurements were obtained with a LEICA M205 C stereomicroscope and are given in mm. Eye diameters were measured as the maximum distance in either dorsal or frontal views. Leg measurements are given as follows: total length (coxa, trochanter, femur, patella, tibia, metatarsus, and tarsus). Terminology follows Wang (2003) and Li et al. (2018b, c). Abbreviations of eyes used in the text are as follows:

ALE
anterior lateral eye; ALE-PLE distance between ALE and PLE; AME anterior median eye; AME-ALE distance between AME and ALE; AME-AME distance between AME and AME; AME-PME distance between AME and PME; PLE posterior lateral eye; PME posterior median eye; PME-PLE distance between PME and PLE; PME-PME distance between PME and PME.

Laboratory protocols and phylogenetic analyses
The DNA barcodes of the putative new species were obtained to test the species boundaries. A partial fragment of the mitochondrial cytochrome oxidase subunit I (CO1) gene was amplified and sequenced using the primers LCO1490-oono (5'-CWACAAAYCATARRGATATTGG-3') and HCO2198-zz (5'-TA-AACTTCCAGGTGACCAAAAAATCA-3'), following Zhao and Li (2017). GenBank accession numbers of CO1 are listed separately in Table 1. The molecular dataset consisted of: CO1 gene, histone 3 (H3) gene, NADH dehydrogenase subunit I (ND1) gene, wingless gene and the ribosomal RNA genes 12S, 16S, 18S, and 28S; in total eight genes of 77 species that were recently published, including 68 species in 33 known genera of Coelotinae (with 27 type species from different genera) as the ingroup, and four species of Ageleninae and Amaurobiidae as the outgroup (Zhao and Li 2017;Okumura and Zhao 2022;, alongside three novel sequences. GenBank accession numbers for all the above genes are shown in Suppl. material 1. Phylogenetic relationships were inferred using both maximum likelihood (ML) and Bayesian inference (BI). First, the best-fit partitioning schemes and models were selected for the RAxML and MrBayes analyses by using Parti-tionFinder v.2.1.1 (Lanfear et al. 2012). ML analysis was conducted in RAxML v.8.0.0 (Stamatakis 2006) using the substitution model GTRCAT for all partitions (partitioned by genes). A rapid bootstrap ('-f a') of 1000 replicate ML inferences was performed to search for the best-scoring ML tree and compute nodal support. BI analysis were performed in MrBayes v.3.2.2 (Ronquist and Huelsenbeck 2003) with posterior distributions estimated by Markov chain Monte Carlo (MCMC) sampling. The appropriate model was selected for each partition gene: the GTR+I+G model was favored for each partition, except that different models were selected for H3 (HKY+I+G), wingless (SYM+I+G) and 18S (K80+I+G). Two simultaneous runs with four MCMC chains were performed for 10 million generations to ensure that the average standard deviation of the split frequency was below 0.01 and to obtain a well-supported consensus tree. Then, ML analysis was also performed in IQ-TREE v.1.6.12 (Nguyen et al. 2015) by using ModelFinder function (-m MFP+MERGE) to select the best-fit model for each partition, and the option '-bb 1000' to estimate the nodal support values.

Results and discussion
The five species of the new genus share similar external genital morphology such as a long femur (more than three times longer than patella), a short patella (c. half the length of tibia), a bent tibia, the base of cymbium with one or two hypophyses, a wide embolus having a widest anterior, a large dorsal apophysis of conductor with a jagged margin; an epigyne lacking epigynal teeth, an anterior atrium located over the swell of epigyne and posterior epigynal sclerite between two swells of the epigyne, spermathecae small (shorter than 1/4 the length of copulatory ducts) and located posteriorly, close to each other, which anterior part fist-like.      Our phylogenetic analyses all infer similar tree topologies (Fig. 1) and strongly support Baiyuerius gen. nov. as a monophyletic clade (ML bootstrap = 100 and 95; BI posterior probability = 1.00). Geographically, species belonging to Baiyuerius gen. nov. are restricted to southern China and northern Vietnam (Fig. 8). Zoogeographic studies suggest that the genus-level distribution of coelotine spiders is regional, and the divergence and formation of these monophyletic genera are closely linked to geological and climatic events that occurred during the Neogene in Eurasia (Zhao and Li 2017;Zhao et al. 2020Zhao et al. , 2022.

Vappolotes
Based on these results, taking into account morphological comparisons, phylogenetic analyses, and zoogeographic considerations, Baiyuerius gen. nov. is established herein. Etymology. The generic name is derived from the pinyin word "Baiyue", referring to the Baiyue region where the new genus is distributed. Baiyue, a loose term dating back to the first millennium BC, was used to denote various populations who inhabited southern China and northern Vietnam. The postfix "-rius" refers to the postfix commonly used in the genera of the Sinodraconarius clade. The gender is masculine.
Diagnosis. The morphological characteristics of Baiyuerius gen. nov. resemble those of Yunguirius, which is the closest genus to Baiyuerius gen. nov., by the dark color of the carapace, endites and labium; tibia longer than patella of male palp; dorsal apophysis of conductor large; embolus thick with swollen base; copulatory ducts membranous, arising posteriorly, along the contour of epigynal atrium. However, it can be distinguished from Yunguirius as follows: 1) the base of cymbium enlarged, with 1 or 2 hypophyses (Figs 3C, 5C, 6C) vs. without any hypophysis in Yunguirius; 2) an atrium located anteriorly and occupying less than or equal to 1/2 of the epigyne ( Description. Medium-sized, total lengths from 8.60 to 11.98. Carapace black turning brown or brown turning yellow-brown, pear-shaped, with longitudinal fovea and darker radial grooves; chelicerae as the same color as the anterior carapace, with three promarginal and two retromarginal teeth; endites and labium dark brown or grey, anteriorly white with black hairs; sternum brown or milk-white, longer than wide. Abdomen yellow-brown, covered with grey hairs, with two pairs of apodemes and four darker chevron-like markings. Spinnerets lighter than sternum in color. Leg formula 4 > 1> 2 > 3. Male palp: femur more than 3 times than patella, patella approx. half of tibia, patellar apophysis thick and enlarged, finger-like, longer than half of tibia and extending over patella, retrolateral tibial apophysis rectangular and lamellar, extending beyond tibia, lateral tibial apophysis of the same shape as patellar apophysis but thinner and shorter, median apophysis spoon-shaped, length of cymbial furrow c. 1/2 length of cymbium, with an enlarged base, the base of cymbium with 1 or 2 hypophyses, embolus widen and slightly elongated, anterior widest, dorsal apophysis of conductor large and in different shapes, in some cases bearing a jagged margin; Female epigyne: epigynal teeth absent, atrium located anteriorly, over the swell of epigyne, internally milk-white, occupying more than or equal to 1/4 of the female epigyne, posterior epigynal sclerite varying in shape and between two swollen parts of epigyne, copulatory ducts beginning at the posterior margin of epigyne, extended anteriorly, copulatory opening located anterolaterally, spermathecae small and located posteriorly, shorter than 1/4 the length of its copulatory ducts, anterior part fist-like, while its base close to each other, fertilization ducts originating from inside of spermathecae. Distribution. Guizhou and Guangdong Province, Guangxi Zhuang Autonomous Region, China and Vinh Phuc Province, Vietnam (Fig. 8). Etymology. The new species is named after the type locality, the Daxi Village; noun in apposition.
Female. Unknown. Distribution. Guangdong Province, China (Fig. 8). Etymology. The new species is named after the type locality, the Tam Dao National Park; noun in apposition.

Baiyuerius zhuping
Female. Unknown. Distribution. Guizhou Province, China (Fig. 8).  Diagnosis. The males of Baiyuerius zuojiang sp. nov. resemble those of B. pindong sp. nov. by the margin of conductor jagged and dorsal apophysis large, patellar apophysis with a blunt and bent distal end; resemble those of B. zhuping sp. nov. by cymbial base with one hypophysis, lateral tibial apophysis finger-like. However, it can be distinguished from them by 1) median apophysis coiled and jagged (Fig. 6B) vs. without any jags and flat (in B. pindong) or with three lobes (in B. zhuping) (Figs 3B, C, 5B, C). The females of B. zuojiang sp. nov. resemble those of B. daxi sp. nov. by glasses-shaped atrium, and copulatory ducts extending first posteriorly then anteriorly, along the sclerotic margins of the atrium, anterior separated from each other. However, it can be distinguished from B. daxi as follows: 1) atrium occupying 1/3 of the epigyne (Fig. 7A) vs. half of epigyne in B. daxi ( Fig. 2A); 2) posterior epigynal sclerite rhomboid, longer than wide (Fig. 7A) vs. pentagonal, as the same length as its width in B. daxi (Fig. 2A); and 3) copulatory ducts originating posteriorly, from the dorsal spermathecae then extending anteriorly (Fig. 7B) vs. originating centrally and near posteriorly, from the ventral spermathecae then extending anteriorly (Fig. 2B).